Biological sensors, and more particularly, fiber optic biological sensors are generally used to determine the presence, absence, and/or concentration of certain chemical and/or molecular constituents occurring in a medium. In practice, a fiber optic type biological sensor is normally used in such applications. That is, in practice, the biological sensor utilizes an optical fiber, having at least a first portion which is immersed into the medium and a second portion which receives light energy. More particularly, the first portion generally receives injected light energy and communicates the received light energy to the fiber portion which is resident or immersed within the medium.
A first chemical and/or molecular constituent which is known to form an affinity complex with the second constituent occurring in the medium is generally and normally directly and/or indirectly attached to the surface of at least the immersed portion of the fiber. The second constituent occurring in the medium is selectively tagged with fluorescent molecules or other molecules which either generate light energy in the presence of an evanescent energy field, or absorb energy from an evanescent field thereby altering the transmission of light through the fiber. As the fiber is immersed into the medium, some amount of the second constituent typically binds with the first constituent.
As light is received into the first portion of the fiber and communicated to the medium immersed fiber portion, an evanescent field is created around the immediate vicinity of the surface of the fiber. This evanescent field causes fluorescent tags occurring in very close proximity to the fiber surface (e.g. only those tags attached to the portions of the second constituent which become actually bound to the fiber surface) to radiate light energy, at some known and predetermined wavelength dependent upon the type and identity of employed tag. This radiated light energy is "tunneled" or communicated into the fiber and subsequently propagated and communicated to a receiver or detector. The amount of such received tunneled light is known to be directly proportional to the amount of the second constituent which binds to the fiber surface. This binding of this second constituent varies either directly or inversely, depending upon experimental design, with the amount or concentration of the second constituent occurring in the medium. In this manner, based upon the intensity of the received evanescently produced light, a calculation is made of the absence, presence, and/or concentration of the second constituent which is occurring in the medium . As previously mentioned other types of energy absorbing molecules may be used to alter the transmission of the tunneled light within the fiber. These absorbing molecules replace the fluorescent tags and represent a different invention embodiment. The concentration or amount of such absorbing molecules in close proximity to the sensor surface (e.g. attached to the second constituent adhering or binding to the first constituent) changes the amount of tunneled light received from the fiber and such change is directly or inversely proportional to the concentration of the second constituent occurring in the medium.
While these biological sensors are generally effective in determining the presence of the second constituent in the medium they suffer from many drawbacks, especially when used to determine the exact medium concentration of the second constituent.
Because such sensors rely upon the specific binding between the fluorescent-tagged constituent and its binding partner at the fiber surface, binding of the fluorescent-tagged constituent to the surface of the fiber in a manner which does not rely upon the specific affinity binding to the binding partner creates a false signal. More particularly, such binding results in fluorescence which is tunneled and communicated to the receiver, but which does not reflect any quantitative relationship between the recognition molecule and its binding partner. This diminishes the accuracy of the sensor. Such binding of molecules to the fiber surface in a manner which does not rely upon specific affinity binding between a recognition molecule (e.g. first constituent) and its partner (e.g. second constituent) is designated as "nonspecific binding". In the case of immunobinding, the antibody is the recognition molecule and the antigen is the partner.
The non-specific binding of non-fluorescent constituents in a biological sample to the surface of the fiber can also adversely affect sensor operation by obscuring the binding partner on the fiber surface thereby interfering with the specific binding between the recognition protein and its ligand on the fiber surface. There is therefore a great need to provide a methodology to reduce and/or eliminate such "nonspecific" binding upon the biological sensor, and there is a further need to provide and to construct a biological sensor upon which constituents do not bind nonspecifically. Such non-specific binding similarly and detrimentally affects binding methodologies using energy absorption molecules in place of the fluorescent tags.
Yet another drawback to these prior biological sensors is that the evanescent field created by injecting light using conventional optical methods is not uniformly generated along the fiber surface and/or is relatively weak in some parts of the sensor surface. This lack of uniformity and weakness prevents some of the bound and tagged constituents from being "counted" or induced to fluoresce, or from absorbing and hence "changing" the evanescent field. Hence, the resulting concentration calculation is incorrect. There is therefore also a need to provide a methodology to increase the strength and/or efficiency of the generated evanescent field and to ensure that the field is substantially and uniformly generated about the fiber. There is also a great need for a biological sensor employing this evanescent field enhancement methodology.
Yet another drawback to these prior optical fiber biological sensors is that the majority of injected light rays traveling within the fiber sensor couple only weakly to the evanescent field at the fiber surface because they do not all impinge on the inside fiber surface at angles (as measured between the fiber surface and the light ray) near to but not greater than the critical angle for the fiber-liquid interface. As a result, the coupling of injected light via the evanescent field to bound and tagged constituents on the fiber surface is less than optimal, the fluorescence stimulated by this evanescent field is reduced, and ultimately the fluorescence from the tagged constituent which is coupled back through the fiber sensor to a fluorescence measuring device is significantly reduced. While techniques such as chemically etching a taper into fiber sensors have been used to concentrate more of the injected light rays into angles close to the critical angle, such techniques have led to fiber sensors which are hard to manufacture, large in size, and which concentrate the injected light only within a section of the fiber sensor.
Yet a further drawback associated with these prior optical fiber biological sensors is that a relatively large amount of the light radiation which is input and output from the sensor is lost due to the nature of the sensor supporting structure and/or to the method by which the fiber optic structure is supported or "held" into the medium of interest.
There is therefore a need to provide a methodology to increase the strength and/or efficiency of the coupling of the injected light to the evanescent field and to insure that the field is substantially and uniformly generated about the sensitized region of the fiber sensor so that small and highly sensitive fiber sensors can be easily manufactured. There is also a great need for a biological sensor employing this evanescent field enhancement methodology. There is also a need to provide a biological fiber optic sensor and sensor support assembly and/or fiber optic structural support methodology which reduces light energy loss associated with the prior art sensor assemblies.